SMAD3 modulates CD4 T-cell proliferation and T-bet expression. (A) One representative histogram showing the CFSE profile of CD4 T cells from WT or SMAD3-KO mice. Fluorescence-activated cell sorter naive CD4 T cells (CD44^loCD62L^hi) were left unstimulated (Ctrl) or were stimulated with anti-CD3ϵ (1 μg/mL) and anti-CD28 (5 μg/mL) in the absence or presence of TGF-β (2.5 ng/mL) for 72 hours. (B) Based on the number of cells in each division peak (as determined by CFSE-labeling intensity), we calculated the number of responding cells, their doubling time, and the number of daughter cells generated per responding cell (4 independent experiments). (C-F) Naive CD4 T cells were cultured under Th1-skewing conditions. (C) The percentage of T-bet⁺ cells was evaluated by intracellular staining and flow cytometric analysis (3-9 independent experiments). (D) One representative staining for T-bet and FOXP3 in CD4 T cells cultured for 72 hours in the presence or absence of TGF-β. (E-F) Mean percentage of CD4⁺ cells expressing T-bet or FOXP3 after culture for 72 hours in the presence of various concentrations of TGF-β (3-5 independent experiments). (G) Percentage of CD4 T cells expressing FOXP3 or RORγt after culture for 72 hours under Treg or Th17-skewing conditions, respectively (4 independent experiments). All histograms represent the mean and SEM. For all comparisons, differences were assessed using a 2-tailed paired Student t test: *P < .05, **P < .01, ***P < .001.